contents Component of plasmid vector bacterial culture Plasmid

contents • Component of plasmid vector • bacterial culture…? ? • Plasmid purification methods • Procedure plasmid DNA prep by Alkaline lysis • Concentration • Determination of DNA

Component of plasmid vector Report : What is f 1 ori. . ? ? Selection marker : ex) Ampr, Tetr, Chlr, Kanr Ex) p. MB 1 low copy number : 15 -20 copies/cell p. UC high copy number : 300 -700 copies/cell

bacterial culture…? ? Bacterial growth curve

Plasmid purification methods • Alkaline lysis Method • Cs. Cl-Et. Br Gradient Equilibium Centrifugation • Anion-Exchange Chromatography Method

Alkaline lysis Method

Cs. Cl-Et. Br Gradient Equilibium Centrifugation 이온구배, 부력에 따라서 gradient 생김 Et. Br 첨가시 Linear DNA – 0. 125 g/cm 3 만큼 밀도 감소 -> 더 많은 부력 얻 음 Supercoiled DNA – 0. 085 g/cm 3 만큼 밀도 감소

Anion-Exchange Chromatography Method

Procedure plasmid DNA prep by Alkaline lysis • Key steps • Materials Buffers and Solutions LB medium – for Cell culture Solution I - resuspension solution Solution II - lysis solution Solution III - Renaturation solution Phenol / Chloroform – for removing protein 100% Et. OH – for precipitating plasmid DNA 70% Et. OH - for removing Salt (washing) TE – for disolving plasmid DNA • Methods

Key steps • Preparation of Cell Culture • Lysis of Cells • Recovery of Plasmid DNA

Materials LB medium – for Cell culture Solution I - resuspension solution 50 m. M glucose 25 m. M Tris-Cl, p. H 8. 0 (p. H 8) 10 m. M EDTA, p. H 8. 0 (p. H 8. 0) Rnase 100μg/ml Solution II - lysis solution 0. 2 N Na. OH 1% (w/v) SDS Solution III – Neutralization solution 5 M potassium acetate 60. 0 ml Glacial acetic acid 11. 5 ml Phenol, Chloroform , isoamylalchohol (25: 24: 1) – for removing protein 100% Et. OH – for precipitating plasmid DNA 70% Et. OH - for removing Salt (washing) TE – for disolving plasmid DNA (20 μg/ml RNase A) 10 m. M Tris-Cl, p. H 8. 0 1 m. M EDTA, p. H 8. 0

Preparation of Cell Culture • Meterials 30 ml of LB medium - for Cell culture Antibiotics transformed bacteria • Procedures 1) Inoculate 30 ml of rich medium containing the appropriate antibiotics with transformed bacteria 2) Culture for overnight at 37℃ with shaking 3) Centrifugate the cultured Cells for collecting at 4000 -5000 rpm for 10 min at 4℃ 4) Discard the supernatant

Lysis of Cells • Meterials Solution I - resuspension solution 50 m. M glucose 25 m. M Tris-Cl, p. H 8. 0 (p. H 8) 10 m. M EDTA, p. H 8. 0 (p. H 8. 0) Rnase 100μg/ml Solution II - lysis solution 0. 2 N Na. OH 1% (w/v) SDS Solution III – Neutralization solution 5 M potassium acetate 60. 0 ml Glacial acetic acid 11. 5 ml • Procedures 1) 2) 3) 4) 5) 6) 7) 8) Resuspend the pellet in 600 μl of Alkaline lysis solution I Sol I: Sol II : Sola III = 1 : 2 : 1. 5 Add 1200 μl of freshly prepared solution II DNA or RNA Mix the contents by gently inverting the tube several times. Protein Incubate the bottle for 3 -5 min at room temperature. Lipid Add 900 μl of ice-cold solution III. Mix the contents gently. Centrifugate the bacterial lysate at 15000 rpm for 30 min at 4 ℃ Collect the supernatant and discard the pellet remaining in the centrifuge bottle.

Recovery of Plasmid DNA • Meterials Isopropanol or 100% Et. OH, 70% Et. OH, TE(p. H 8. 0) • Procedures 1) Measure the volume of the supernatant. 2) Transfer the supernatant with 0. 6 volume of iso propanol to a fresh bottle and gently mix. 3) Centrifuge for 15 min at 15000 rpm 4) remove the supernatant fluid and rinse the pellet with 1 ml of ice cold 70% Et. OH 5) Centrifuge for 5 min at 15000 rpm 6) Remove the Et. OH 7) Air dry the pellet but not overdry 8) Dissolve the pellet of DNA in 100 μl of TE(p. H 8. 0)

Ethanol precipitation of DNA • Definition Precipitation of Nucleic acid molecules by ethanol plus salt. Used primarily as a means of concentrating DNA or RNA from aqueous solutions. • Principle 1) Counter-ions(cation) such as Na+ bind to the charged groups and reduce repulsive forces between the polynucleotide chains to the point where a precipitate can form 2) Then, the nucleotide bound with counter-ions can be separated from ethanol and precipitated in the bottom of tube. • Cation • 2 vol of Et. OH or 0. 62 -1 vol of Isopropanol Salt Ammonium acetate Lithium chloride Sodium Chloride Sodium acetate Stock solution(M) 10 8 5 3 (p. H 5. 2) Final conc. (M) 2 -2. 5 0. 8 0. 2 0. 3

Concentration and Determination of DNA High quality DNA OD 260(DNA) / OD 280(Protein) = 1. 8 -2. 0 1. 8미만일 경우-단백질이나 phenol존재 농도계산: 흡광도 측정 값 ⅹ희석비율(%)ⅹDNA측정 상수 값 (50) or RNA측정 상수 값 (40)/단위 ㎕로 환산 Ex) DNA를 1/100으로 dilution OD 260 = 1. 6 x 50 ng/μl x 100 = 8000 ng/ μl or 8 μg/ μl